Single Nucleotide Polymorphism Profiles Research Articles

Genomic assessment of white clover and perennial ryegrass genetic resources

Forage resources conserved in genebanks, such as the Margot Forde Germplasm Centre (MFGC; PalmerstonNorth), are reservoirs of genetic diversity important for the development of cultivars adapted to abiotic stresses and environmental constraints. Genomic tools, including genotyping-by-sequencing (GBS), can support identification of manageable subsets (core collections) that are genetically representative of these large germplasm collections, for phenotypic characterisation. We used GBS to generate SNP (single nucleotide polymorphism) profiles for 172 white clover (WC) and 357 perennial ryegrass (PRG) MFGC-sourced accessions and estimated genetic relationships amongst accessions. In WC, accessions aligned along an east-west transect from Kazakhstan to Spain, identifying major diversity in Caucasus/Central Asia and Iberian Peninsula. A key feature was the reduced diversity present in New Zealand (NZL) accessions. Similarly, for PRG, most NZL accessions coalesced as one group, distinct from large clusters associated with the Iberian Peninsula, Italy and eastern Mediterranean/Caucasian region. These results emphasise the relatively narrow genetic diversity in NZL WC and PRG, and the broad extent of largely unexploited global diversity. Capturing global genetic variation incore collections will support pre-breeding programmes to mobilise novel genetic variation into New Zealand-adapted genetic backgrounds, enabling development of cultivars with non-traditional traits including enhancedclimate resilience and environmental performance.

Abstract 1212: Quantitative ancestry analysis in Hispanic uveal melanoma patients reveals novel disease susceptibility loci

Abstract Uveal Melanoma (UM) is the most common eye cancer in adults and leads to metastatic death in up to half of patients. The molecular landscape of UM has been extensively characterized, and this information is now widely used in clinical management. Research interest in UM has expanded dramatically in the past decade, with landmark genomic studies highlighting its unique and deadly clinical and molecular characteristics however this pool of genetic data has been performed almost entirely in white non-Hispanic patients of European ancestry. Hispanics are the second most common ethnic group affected by UM, and one of the fastest growing ethnic groups in the U.S. Individuals who self-identify as Hispanic. This study included 141 patients with posterior uveal melanomas, 72 who self identified as White non-Hispanic, 61 as Hispanic, 4 as Black non-Hispanic, 2 as Asian non-Hispanic, and 2 as Other/Multiracial non-Hispanic. In order to by-pass ethnic and cultural variables that influence self reporting demographic information, global and local ancestry was assessed. Leukocyte DNA from each patient was genotyped, generating whole genome single nucleotide polymorphism (SNP) profiles. Quality control was performed keeping only bi allelic SNPs and SNP call rate of >98%. Global ancestral estimates, using greater than 100,000 SNPs, were calculated against reference samples from West African, East Asian, European, and Native American ancestral populations. ADMIXTURE was used to estimate the ancestral proportions among the UM patients. Greater than 500,000 SNPs were phased and local ancestral calls were calculated on each patient. The genetic structure of individual patient samples revealed a diverse ancestral admixture among self-reported Hispanic patients with UM. Significant differences in global European ancestry were shown between Class 1A and Class 1B patients, where Class 1B patients were more European (P = 0.0087). Kruskal-Wallis test showed a significant difference between the three groups' european ancestry showing that Class 1A, Class 1B, and Class 2 distributions are not all the same (P=0.035). Patients with higher European ancestry are also more likely to be PRAME-positive (P=0.02). For local ancestry analysis, a linear regression was performed at each haplotype. A significance threshold of 0.000694 was established after correction for the mean number of ancestral switches in the cohort. Two regions on chr20 reached genome wide significance (P<0.0006) with an enrichment of European ancestry in more aggressive malignancies. These findings present an exciting opportunity for studying ancestral genetic determinants on patient outcome in UM. The results show that genetic ancestry may influence the occurrence and clinical significance of prognostic biomarkers and patient outcomes in UM. Utilizing genetic ancestry may aid in our understanding and guide future management of ocular pathologies in admixed individuals. Citation Format: Daniel Alexander Rodriguez, Margaret I. Sanchez, Christina L. Decatur, J. William Harbour. Quantitative ancestry analysis in Hispanic uveal melanoma patients reveals novel disease susceptibility loci [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1212.

Post-hoc analysis of single nucleotide polymorphism profile for eyes with vascularized pigment epithelial detachment due to ARMD.

This post-hoc case-control study compares single nucleotide polymorphism (SNP) profile of eyes with vascularized pigment epithelial detachment (vPED) due to age-related macular degeneration (ARMD) with: (1) Control-1 eyes (no ARMD and AREDS Severity Scale 0); and (2) Control-2 eyes (drusen or AREDS Severity Scale 2). SNP profile of High Responders (HR) was also compared with Low Responders (LR) to ranibizumab. Blood samples from 40 patients with vPED treated with ranibizumab were sent for SNP-specific genotype analysis for comparison of variant allele frequencies of 23 SNPs associated with ARMD (VAF) to VAF in 184 Control-1 eyes, and VAF in 85 Control-2 eyes. VAF of HR-50 (⩾50% decrease in PED height) and VAF of HR-75 (⩾75% decrease in PED height) were also compared with VAF of LR. These SNPs were more frequent in vPED than Control-1 eyes: APOE rs4420638 (A/G), HTRA1 rs104904924 (G/T), VEGF rs943080 (T/C), CFH rs1061170 (T/C), CFH rs2274700 (C/T), CFH rs10737680 (A/C), CFH rs10801555 (G/A). These SNPs were more frequent in vPED than Control-2 eyes: APOE rs4420638 (A/G), CFI rs4698775 (G/T), COL15A1/TGFBR1 rs334353 (T/G). FRK rs3812111 (T/A) was more frequent in HR-50 and HR-75 eyes compared with LR. Seven SNPs were more frequent in vPED eyes than non-ARMD eyes, and three SNPs were more frequent in vPED eyes than drusen eyes. Adjusting for multiplicity, only CFH rs2274700 (C/T) was significant for first comparison, and only COL15A1/TGFBR1 rs334353 (T/G) was significant for second comparison. APOE rs4420638 (A/G) was the single SNP more frequently linked to vPED eyes for both comparisons. FRK rs3812111 (T/A) was consistently associated with high responders.

TERC Variants Associated with Short Leukocyte Telomeres: Implication of Higher Early Life Leukocyte Telomere Attrition as Assessed by the Blood-and-Muscle Model.

Short leukocyte telomere length (LTL) is associated with atherosclerotic cardiovascular disease (ASCVD). Mendelian randomisation studies, using single nucleotide polymorphisms (SNPs) associated with short LTL, infer a causal role of LTL in ASCVD. Recent results, using the blood-and-muscle model, indicate that higher early life LTL attrition, as estimated by the ratio between LTL and skeletal muscle telomere length (MTL), rather than short LTL at conception, as estimated by MTL, should be responsible of the ASCVD-LTL connection. We combined LTL and MTL measurements and SNPs profiling in 402 individuals to determine if 15 SNPs classically described as associated with short LTL at adult age were rather responsible for higher LTL attrition during early life than for shorter LTL at birth. Two of these SNPs (rs12696304 and rs10936599) were associated with LTL in our cohort (p = 0.027 and p = 0.025, respectively). These SNPs, both located on the TERC gene, were associated with the LTL/MTL ratio (p = 0.007 and p = 0.037, respectively), but not with MTL (p = 0.78 and p = 0.32 respectively). These results suggest that SNPs located on genes coding for telomere maintenance proteins may contribute to a higher LTL attrition during the highly replicative first years of life and have an impact later on the development of ASCVD.

Age-Related Changes in Hair Shaft Protein Profiling and Genetically Variant Peptides

Recent reports highlight possible improvements in individual identification using proteomic information from human hair evidence. These reports have stimulated investigation of parameters that affect the utility of proteomic information. In addition to variables already studied relating to processing technique and anatomic origin of hair shafts, an important variable is hair ageing. Present work focuses on the effect of age on protein profiling and analysis of genetically variant peptides (GVPs). Hair protein profiles may be affected by developmental and physiological changes with age of the donor, exposure to different environmental conditions and intrinsic processes, including during storage. First, to explore whether general trends were evident in the population at different ages, hair samples were analyzed from groups of different subjects in their 20’s, 40’s and 60’s. No significant differences were seen as a function of age, but consistent differences were evident between European American and African American hair profiles. Second, samples collected from single individuals at different ages were analyzed. Mostly, these showed few protein expression level differences over periods of 10 years or less, but samples from subjects at 44 and 65 year intervals were distinctly different in profile. The results indicate that use of protein profiling for personal identification, if practical, would be limited to decadal time intervals. Moreover, batch effects were clearly evident in samples processed by different staff. To investigate the contribution of storage (at room temperature) in affecting the outcomes, the same proteomic digests were analyzed for GVPs. In samples stored over 10 years, GVPs were reduced in number in parallel with the yield of identified proteins and unique peptides. However, a very different picture emerged with respect to personal identification. Numbers of GVPs sufficed to distinguish individuals despite the age differences of the samples. As a practical matter, three hair samples per person provided nearly the maximal number obtained from 5 or 6 samples. The random match probability (where the log increased in proportion to the number of GVPs) reached as high as 1 in 108. The data indicate that GVP results are dependent on the single nucleotide polymorphism profile of the donor genome, where environmental/processing factors affect only the yield, and thus are consistent despite the ages of the donors and samples and batchwise effects in processing. This conclusion is critical for application to casework where the samples may be in storage for long periods and used to match samples recently collected.

Single nucleotide polymorphisms (SNPs) as predictors of efficacy of cabazitaxel in patients with metastatic castration-resistant prostate cancer (mCRPC).

e17582 Background: Cabazitaxel is a semi-synthetic derivative of a natural taxoid approved for the treatment of mCRPC patients (pts) after failure to docetaxel. Despite its proven efficacy, there is variability in the response, progression-free survival (PFS) and overall survival (OS) of pts. Changes in the genetic constitution of the individual such as the SNPs could explain this variability. The aim of this study was to evaluate the impact of certain SNPs in cabazitaxel activity. Methods: Clinical data from 67 mCRPC pts treated with cabazitaxel between March 2011 and October 2016 were collected. DNA was isolated from formalin fixed paraffin-embedded tumor samples. 56 SNPs in 5 genes related with metabolism and/or mechanism of action of cabazitaxel (CYP3A4, CYP3A5, ABCB1, TUBB1, CYP2C8) were chosen based on their Minor Allele Frequency, linkage disequilibrium and information from dbSNP and analyzed by TaqMan OpenArray (Lifetech). The presence/absence of mutant alleles of the selected SNPs was correlated with clinical features, progression free survival (PFS) and overall survival (OS) of prostate cancer. Chi-square test and Kaplan-Meier with log-rank test were used for statistical analyses. Results: The median age was 61 years (range 44-82). 56.7% (n = 38) had a Gleason score ≥8 and 94% had received docetaxel in first line. Type of response to cabazitaxel was associated with median OS (Partial response = 24.35 months, Stable disease = 11.16 months, Progression disease = 5.8 months; p= 0.045). Univariate analysis, showed worsed OS at 1 year for wild type status of SNP rs151352 (OR = 4, 95%CI 1.27-12.58, p= 0.029). In addition, two SNPs (rs11773597, rs1202186) were associated with radiological response to cabazitaxel ( p= 0.031 and p= 0.030 respectively). Other 7 SNPs (rs11773597, rs2235040, rs1045642, rs1419745, rs1202170, rs6949448, rs11572093) were associated ( p<0.05) with Gleason score, pain, PSA doubling time, febrile neutropenia and asthenia. Conclusions: A particular SNP profile could be predictive of efficacy and related with toxicity in mCRPC population treated with cabazitaxel after progression to docetaxel. These outcomes become particularly relevant in patient selection given the recent results of the CARD trial.

On the origin of European sheep as revealed by the diversity of the Balkan breeds and by optimizing population-genetic analysis tools

BackgroundIn the Neolithic, domestic sheep migrated into Europe and subsequently spread in westerly and northwesterly directions. Reconstruction of these migrations and subsequent genetic events requires a more detailed characterization of the current phylogeographic differentiation.ResultsWe collected 50 K single nucleotide polymorphism (SNP) profiles of Balkan sheep that are currently found near the major Neolithic point of entry into Europe, and combined these data with published genotypes from southwest-Asian, Mediterranean, central-European and north-European sheep and from Asian and European mouflons. We detected clines, ancestral components and admixture by using variants of common analysis tools: geography-informative supervised principal component analysis (PCA), breed-specific admixture analysis, across-breed f_{4} profiles and phylogenetic analysis of regional pools of breeds. The regional Balkan sheep populations exhibit considerable genetic overlap, but are clearly distinct from the breeds in surrounding regions. The Asian mouflon did not influence the differentiation of the European domestic sheep and is only distantly related to present-day sheep, including those from Iran where the mouflons were sampled. We demonstrate the occurrence, from southeast to northwest Europe, of a continuously increasing ancestral component of up to 20% contributed by the European mouflon, which is assumed to descend from the original Neolithic domesticates. The overall patterns indicate that the Balkan region and Italy served as post-domestication migration hubs, from which wool sheep reached Spain and north Italy with subsequent migrations northwards. The documented dispersal of Tarentine wool sheep during the Roman period may have been part of this process. Our results also reproduce the documented 18th century admixture of Spanish Merino sheep into several central-European breeds.ConclusionsOur results contribute to a better understanding of the events that have created the present diversity pattern, which is relevant for the management of the genetic resources represented by the European sheep population.

Transmission Genetics of a Sorghum bicolor × S. halepense Backcross Populations.

Despite a “ploidy barrier,” interspecific crosses to wild and/or cultivated sorghum (Sorghum bicolor, 2n = 2x = 20) may have aided the spread across six continents of Sorghum halepense, also exemplifying risks of “transgene escape” from crops that could make weeds more difficult to control. Genetic maps of two BC1F1 populations derived from crosses of S. bicolor (sorghum) and S. halepense with totals of 722 and 795 single nucleotide polymorphism (SNP) markers span 37 and 35 linkage groups, with 2–6 for each of the 10 basic sorghum chromosomes due to fragments covering different chromosomal portions or independent segregation from different S. halepense homologs. Segregation distortion favored S. halepense alleles on chromosomes 2 (1.06–4.68 Mb, near a fertility restoration gene), 7 (1.20–6.16 Mb), 8 (1.81–5.33 Mb, associated with gene conversion), and 9 (47.5–50.1 Mb); and S. bicolor alleles on chromosome 6 (0–40 Mb), which contains both a large heterochromatin block and the Ma1 gene. Regions of the S. halepense genome that are recalcitrant to gene flow from sorghum might be exploited as part a multi-component system to reduce the likelihood of spread of transgenes or other modified genes. Its SNP profile suggests that chromosome segments from its respective progenitors S. bicolor and Sorghum propinquum have extensively recombined in S. halepense. This study reveals genomic regions that might discourage crop-to-weed gene escape, and provides a foundation for marker-trait association analysis to determine the genetic control of traits contributing to weediness, invasiveness, and perenniality of S. halepense.

Genomic Investigation Reveals Contaminated Detergent as the Source of an Extended-Spectrum-β-Lactamase-Producing Klebsiella michiganensis Outbreak in a Neonatal Unit.

Klebsiella species are problematic pathogens in neonatal units and may cause outbreaks, for which the sources of transmission may be challenging to elucidate. We describe the use of whole-genome sequencing (WGS) to investigate environmental sources of transmission during an outbreak of extended-spectrum-β-lactamase (ESBL)-producing Klebsiella michiganensis colonizing neonates. Ceftriaxone-resistant Klebsiella spp. isolated from neonates (or their mothers) and the hospital environment were included. Short-read sequencing (Illumina) and long-read sequencing (MinION; Oxford Nanopore Technologies) were used to confirm species taxonomy, to identify antimicrobial resistance genes, and to determine phylogenetic relationships using single-nucleotide polymorphism profiling. A total of 21 organisms (10 patient-derived isolates and 11 environmental isolates) were sequenced. Standard laboratory methods identified the outbreak strain as an ESBL-producing Klebsiella oxytoca, but taxonomic assignment from WGS data suggested closer identity to Klebsiella michiganensis Strains isolated from multiple detergent-dispensing bottles were either identical or closely related by single-nucleotide polymorphism comparison. Detergent bottles contaminated by K. michiganensis had been used for washing milk expression equipment. No new cases were identified once the detergent bottles were removed. Environmental reservoirs may be an important source in outbreaks of multidrug-resistant organisms. WGS, in conjunction with traditional epidemiological investigation, can be instrumental in revealing routes of transmission and guiding infection control responses.

Association between single nucleotide polymorphism rs1044925 in the acyl‐CoA:cholesterol acyltransferase‐1 gene and plasma lipid parameters in ischemic stroke subjects

Acyl‐coenzyme A: cholesterol acyltransferase‐1 (ACAT‐1) is an intracellular enzyme that catalyzes the synthesis of cholesteryl esters from long‐chain fatty acyl‐CoA and cholesterol in various tissues and plays a major role in cellular cholesterol homeostasis and in atherosclerosis. The present study was carried out to clarify the association of ACAT‐1 rs1044925 single nucleotide polymorphism (SNP) and plasma lipid profiles and lipoproteins levels in patients with ischemic stroke. A total of 100 ischemic stroke patients and 100 controls matched for sex and aged 46–87 were selected. Plasma lipid profiles were measured and lipoprotein ratios were calculated using Excel software. Genotyping of the of the ACAT‐1 rs1044925 SNP was performed by polymerase chain reaction and restriction fragment length polymorphism (PCR‐RFLP) combined with 2% gel electrophoresis and then confirmed by direct sequencing. There were significant difference (P<0.05) in the genotypic and allelic frequencies of ACAT‐1 rs1044925 SNP between the stroke and normolipidemic subjects. The frequencies of AA, AC and CC genotypes of the ACAT‐1 gene were 64%, 33% and 3% for the control and 38%, 57% and 5% for the stroke subjects respectively. The frequencies of A and C alleles were 80.50% and 19.50% for the control and 66.50% and 33.50% for the stroke subjects (P < 0.0001) respectively. The association of genotypes and plasma lipid profiles and lipoprotein ratios were found for both subjects. The A allele carriers had lower plasma TC, TG, VLDL‐C, ApoB levels and other lipid parameters as compared to the C allele carriers for both subjects. The C allele carriers of ACAT‐1 rs1044925 SNP were responsible for increased dyslipidemia and lipid parameters for both subjects. The results of this study suggest that the polymorphism of rs1044925 in the ACAT‐1 gene is associated with plasma lipid profiles and lipoprotein ratios in patients with ischemic stroke.

Molecular typing of Cyclospora cayetanensis in produce and clinical samples using targeted enrichment of complete mitochondrial genomes and next-generation sequencing

BackgroundOutbreaks of cyclosporiasis, a diarrheal illness caused by Cyclospora cayetanensis, have been a public health issue in the USA since the mid 1990’s. In 2018, 2299 domestically acquired cases of cyclosporiasis were reported in the USA as a result of multiple large outbreaks linked to different fresh produce commodities. Outbreak investigations are hindered by the absence of standardized molecular epidemiological tools for C. cayetanensis. For other apicomplexan coccidian parasites, multicopy organellar DNA such as mitochondrial genomes have been used for detection and molecular typing.MethodsWe developed a workflow to obtain complete mitochondrial genome sequences from cilantro samples and clinical samples for typing of C. cayetanensis isolates. The 6.3 kb long C. cayetanensis mitochondrial genome was amplified by PCR in four overlapping amplicons from genomic DNA extracted from cilantro, seeded with oocysts, and from stool samples positive for C. cayetanensis by diagnostic methods. DNA sequence libraries of pooled amplicons were prepared and sequenced via next-generation sequencing (NGS). Sequence reads were assembled using a custom bioinformatics pipeline.ResultsThis approach allowed us to sequence complete mitochondrial genomes from the samples studied. Sequence alterations, such as single nucleotide polymorphism (SNP) profiles and insertion and deletions (InDels), in mitochondrial genomes of 24 stool samples from patients with cyclosporiasis diagnosed in 2014, exhibited discriminatory power. The cluster dendrogram that was created based on distance matrices of the complete mitochondrial genome sequences, indicated distinct strain-level diversity among the 2014 C. cayetanensis outbreak isolates analyzed in this study.ConclusionsOur results suggest that genomic analyses of mitochondrial genome sequences may help to link outbreak cases to the source.

DOP54 Integrated network analysis using patient-specific single-nucleotide polymorphism profiles uncovers new pathways involved in ulcerative colitis pathogenesis

Abstract Background Genome-wide association studies have deciphered the single nucleotide polymorphisms (SNPs) which are responsible for ulcerative colitis (UC) susceptibility. However, to understand how these SNPs are involved in UC, additional methods are necessary. One such approach is in silico network propagation modelling, which can discover how the effects of SNPs in UC can affect the whole cell. A complementary approach is weighted gene co-expression network analysis (WGCNA), where co-regulated genes are identified using transcriptomic data. Integrating these two methods can shed light on how SNPs are affecting the transcriptome of UC patients. Methods We used immunochip profiles of 941 UC patients and focussed on UC-associated SNPs altering regulatory regions. Based on these regions, we identified affected genes. To understand how their corresponding proteins rewire transcriptional regulation, we predicted the path between these proteins and relevant transcription factors (TF) using the OmniPath signalling network (http://omnipathdb.org). From the TFs, we propagated the signal further to target genes using TFlink (https://tflink.net) and GTRD (http://gtrd.biouml.org). To evaluate the predicted network propagation signal, we conducted WGCNA with transcriptomics data from 46 matching patients’ (GEO ID: GSE48959). To interpret the results, we used Gene Ontology Biological Process annotations of the target genes, and we compared the function and regulation of affected genes and the determined WGCNA modules. Results We found 9 predominant signalling pathways, some already known from other studies to be involved in UC pathogenesis, including NFkB signalling, chemokine signalling, Notch pathway, JAK/STAT signalling. Downstream of these pathways we identified potential key TFs regulate the UC phenotype, for example NFKB1, GATA3, GTF2I. The targets of these TFs were enriched in the WGCNA modules of the patients. The WGCNA modules and the transcriptionally affected genes had enriched processes including cell migration, TGF-β signalling, exocytosis, adaptive T- and B-cell-specific immune responses and tight junctions. We also found myogenetic development specific TFs affected transcriptionally such as MyoD, MEF2A, MEF2D. We are currently validating these results through patient-specific biopsies. Conclusion In silico methods bring us closer to understanding UC pathogenesis. Our results suggest that in a well-defined set of patients, weakened tight junctions and insufficient immune response can lead to dysfunctional epithelial barrier, resulting in poor wound healing in UC. We hope the developed workflow will provide novel diagnostic and therapeutic options in UC.

Identifying geographic origins of the Escherichia coli isolates from food by a method based on single-nucleotide polymorphisms

BackgroundE.coli is an important foodborne pathogen. Rapid and robust tracking of the source of E. coli is the key step to control foodborne infections. ResultsIn this study, a genotyping and tracing method based on highly discriminatory single nucleotide polymorphisms (SNPs) was developed to investigate the geographical origin of E. coli in food. A highly informative set of 12 SNPs was derived from 4 housekeeping genes in E. coli multilocus sequence typing (MLST) database. A collection of 253 E. coli isolates from food in 12 countries and regions were screened, resulting in a total of 61 profiles, 35 geographically specific SNP profiles were revealed and further verified by blind sample test. Also, the evolutionary relationship of 61 SNP profiles with different geographical origins was established by the enhanced analysis Based Upon Related Sequence Types (eBURST) analysis, which provided evidence that strains of different geographical origins owned the same ancestor strain. ConclusionsOur study established a powerful method based on a set of 12 SNPs for identifying geographical origins. The blind sample analysis proved that this SNPs panel had a high traceability of E. coli in food. Furthermore, this method based on SNPs combined with eBURST analysis revealed the potential evolutionary relationship between E.coli strains of different geographical origins.

RNA-seq based transcriptome profiling provides important insights into progression of gastric cancer

Abstract Background Gastric cancer (GC), ranking fifth among cancers, is the third most common cause of cancer related mortality in the world. The symptoms of GC occur rather in the late stages, making early diagnosis a challenging task to clinicians compromising disease management. Therefore, efforts are underway to develop effective biomarkers for early diagnosis of the disease and to identify potential targets for drug development. Methods Illumina RNA-Seq based transcriptome profiles of six tumor and normal tissue pairs at different stages (stage I, II and III) of GC were generated to identify differentially expressed genes (DEGs) and single nucleotide polymorphisms (SNPs) implicated in gastric tumorigenesis. The major clusters of candidate genes at different stages of GC and associated enriched gene ontology terms were analysed. Transcription factors involved in GC were also identified and the protein-protein interaction networks were constructed. Results A total of 2207 differentially expressed genes including 972 upregulated genes and 1235 downregulated genes were identified covering stage I, II and III of disease progression. The SNP profiles revealed gene enrichment in cancer related pathways including apoptosis, mTOR and MAPK signalling. The DEGs were accommodated in various ontology categories primarily with digestion system and digestive tract development processes. Functional enrichment of SNPs showed GO categories such as immune system process, regulation of signalling, response to stress, transport, etc. Furthermore, 18 upregulated and 21 downregulated transcription factors were identified during cancer progression. Conclusion Stage-specific identification of DEGs and transcription factors may help in the better understanding of the molecular pathogenesis of gastric cancer. Our findings will also provide useful leads for developing future strategies for the management of gastric cancer. Legal entity responsible for the study The author. Funding Defence Institute of Physiology & Allied Sciences (DIPAS), Defence Research and Development Organisation (DRDO). Disclosure The author has declared no conflicts of interest.

Abstract GMM-021: UTILIZATION OF PATIENT-DERIVED TUMOR XENOGRAFT (PDX) MODELS IN ONCOLOGY

Abstract BACKGROUND: The 5-year survival rate of patients with recurrent epithelial ovarian cancer is only 30%, in part due to relapse and resistance to first-line platinum-based chemotherapy. Even with marked progress toward understanding ovarian cancer biology, the translation of research findings into new therapies is still an enormous barrier to progress. Recent data suggests a 90% failure rate for new oncology drugs in the clinic. Development and preclinical testing of new cancer therapies has been limited by the availability of clinically relevant models that recapitulate the molecular and phenotypic characteristics of primary ovarian cancers. To overcome this barrier, we have initiated collaborative project between Stephenson Cancer Center (SCC) and Oklahoma Medical Research Foundation (OMRF) with the goal to generate a biobank of patient-derived xenograft (PDX) models derived directly from ovarian cancer patients undergoing therapy. RESULTS: PDX tumor models are generated in the Patient-Derived Xenograft and Preclinical Therapeutics (PDX-PCT) Core at OMRF and used to improve preclinical evaluation of new drugs towards more personalized medicine. We have collected blood samples and tumor tissues from consenting patients having primary debulking surgery at Stephenson Cancer Center since 2015 (>140 unique patients). Fresh tumor tissue is minced into small tumor chunks (~2 mm) and implanted into immunocompromised mice. PDXs are expanded for 3-4 passages and characterized based on tumor type, histology and molecular characteristics. Immunohistochemical and molecular analysis showed that histology of the original patient tumors as well as the expression of commonly used markers for high-grade serous ovarian cancer such as cytokeratin, PAX8 and WT1 are perfectly maintained in the derivative PDX lines. Genotyping of patients' tumors and their corresponding PDXs using Illumina's Exome Array revealed that single nucleotide polymorphism (SNP) profile found in the original tumors is perfectly maintained in PDX lines. One of the PDX limitations is that human tumor stromal and immune cells are being replaced with mouse equivalents upon in vivo propagation of PDXs, which negatively affects quality of genomic or proteomic analyses. To overcome this challenge, we optimized a method to deplete mouse cells from PDX tumors by magnetic cells separation. Our data showed that ovarian cancer PDXs contain around 28-54% of human tumor cells, where the rest are mouse cells. We also optimized protocols to utilize pure population of human tumor cells isolated from PDXs to transduce these with lentiviral vectors expressing GFP and/or luciferase to generate luciferized PDXs. Luciferized ovarian PDXs are excellent models used for non-invasive orthotopic tumor growth and drug response monitoring. CONCLUSIONS: Collection of our PDXs illustrates heterogeneity and diversity of human ovarian tumors. However, each PDX maintains essential molecular features of the original patients' tumor. Our goal is to generate clinically faithful ovarian tumor models that will provide a platform to learn more about cancer biology and to screen these for new better therapies. Citation Format: Magdalena Cybula, Katherine Moxley, Lin Wang, Luyao Wang, Magdalena Bieniasz. UTILIZATION OF PATIENT-DERIVED TUMOR XENOGRAFT (PDX) MODELS IN ONCOLOGY [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-021.

Profiling SNP and Nucleotide Diversity to Characterize Mekong Delta Rice Landraces in Southeast Asian Populations.

Single nucleotide polymorphism (SNP) analyses are a powerful tool to examine structure of local rice population. 3000 dataset of IRRI facilitates SNP profiling of Southeast Asian rice populations. Mekong Delta population is featured by comparisons with the other populations. The low π-value SNPs well-profile unique genetic regions in their genomes. Recent analyses using single nucleotide polymorphism (SNP) are a feasible mean for local collections which potentially possess useful, but not large, genetic variations. Genomic sequences of more than 3000 accessions released by the International Rice Research Institute (IRRI) can be used to characterize various local rice (Oryza sativa) populations. The aim of this study was to develop a method to facilitate genomic characterization of local rice populations. We mainly used 99 indica rice accessions (81 landraces and 18 improved varieties) from the Mekong Delta Development Research Institute (MDI). We obtained 2301 SNPs after a genomic sequencing analysis of the 99 rice accessions and subsequent filtering. Within the IRRI's dataset, the landraces fell into a cluster consisting of accessions from Southeast Asian countries (Ind3 cluster), and the MDI improved varieties were grouped in a cluster containing IRRI improved varieties (Ind1B cluster). A principal component analysis suggested that geographical location strongly affects phylogenetic relationships, and the MDI landraces were placed into a Vietnam+Cambodia group. To detect the nucleotide diversity within a population, π-value is commonly used. We think that whole genome distribution of π-values representing the nucleotide diversity of each population can be used to characterize local populations. Our simple profiling using low π-value genomic regions was able to reveal regional characteristics of rice genomes and should be useful for identifying local rice populations.

Primary ciliary dyskinesia gene contribution in Tunisia: Identification of a major Mediterranean allele.

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disease of motile cilia. Even though PCD is widely studied, North-African patients have been rarely explored. In this study, we aim at confirming the clinical diagnosis and explore the genetic spectrum of PCD in a cohort of Tunisian patients. Forty clinically diagnosed patients with PCD belonging to 34 families were recruited from Tunisian pediatric departments. In each proband, targeted capture PCD panel sequencing of the 40 PCD genes was performed. PCD panel sequencing identified bi-allelic mutations in 82% of the families in eight PCD genes. Remarkably, 23.5% of patients carried the same c.2190del CCDC39 mutation. Single nucleotide polymorphism profiling in six unrelated patients carrying this mutation has revealed a founder effect in North-African patients. This mutation is estimated to date back at least 1,400-1,750 years ago. The identification of this major allele allowed us to suggest a cost-effective genetic diagnostic strategy in North-African patients with PCD.

Optimizing DNA recovery and forensic typing of degraded blood and dental remains using a specialized extraction method, comprehensive qPCR sample characterization, and massively parallel sequencing

Human dental remains encountered in criminal casework evidence, missing person cases, or mass disaster tragedies provide a valuable sample source for DNA typing when suitable soft tissue is unavailable. Using traditional methods, teeth samples can be challenging to process, resulting in low-quantity and/or quality nuclear DNA and insufficient profiles for comparisons. This study examines the performance of a three-part nuclear DNA analysis workflow for teeth samples based on (1) improved dental tissue recovery using the Dental Forensic Kit (DFKMR) (Universidad de los Andes) and DNA extraction with QuickExtract™ FFPE DNA Extraction Kit (Lucigen®), (2) quantification with InnoQuant® HY (InnoGenomics Technologies) for sensitive assessment of total human and male DNA quantity/quality, and (3) massively parallel sequencing for simultaneous genotyping of 231 short tandem repeat (STR) and single-nucleotide polymorphism (SNP) markers with the ForenSeq® DNA Signature Prep Kit (Verogen, Inc.). Initial evaluation of artificially degraded blood samples (n = 10) achieved highly sensitive and informative quantification results with InnoQuant® HY, enabling successful first pass genotyping with the MiSeq FGx® System. Twenty-three STR alleles (out of 85) and 70 identity informative SNP loci (out of 94) were recovered from two pg total long target DNA input (0.86 ng total short target input) and an InnoQuant degradation index (DI) of 460 (severely degraded). The three-part workflow was subsequently applied to teeth samples (dental pulp, root cement tissues; n = 13) with postmortem intervals (PMI) of the teeth ranging from 8 days to approximately 6 months. Informative SNP and STR DNA profiles were obtained, to include 78 STR alleles and 85 identity informative SNP loci typed (of 94 total SNP targets) in a 1 month, four-day PMI root cement sample with one pg total long target DNA input and a DI of 76. These data indicate successful performance of the proposed workflow from degraded DNA from teeth samples.

Development of DNA barcode for rapid identification of Epimedium elatum (Morren & Decne) from Northwestern Himalayas in India

Epimedium elatum (Morren & Decne) is a rare monotypic high altitude medicinal species, endemic to shady forests in Northwestern Himalayas, India. Its recent chemoprofiling has revealed high concentration of Epimedin B, C & Icariin, thus matching quality standards of Herba Epimedii. DNA barcode was hitherto unreported in E.elatum. Three plastid genes (matK, rbcL & trnH-psbA) and one nuclear region gene (ITS) were partially sequenced to develop unique barcode and also investigate nucleotide diversity and phylogenetic relationship among five geographically diverse accessions of E.elatum. We used the Kimura 2-parameter (K2P) evolutionary model to calculate basic sequence statistics like conserved, variable, parsimony informative and singleton sites, including pairwise genetic distance in MEGA X software. Besides, we employed a maximum likelihood phylogenetic tree method for estimating the evolutionary divergences. The present study revealed correct blast validation of E.elatum populations with other Epimedium species. A total of 18 new sequences were obtained and submitted to NCBI Genbank via BankIT submission tool (MH615782–MH615799). Highest variable (polymorphic) sites were found in matK (269) followed by ITS (115); rbcL (18) and least in trnH-psbA (16). The evolutionary sequence divergence varied from 0.01 in rbcL/trnH-psbA; 0.09 in ITS to 0.19 in matK. Haplotype and single nucleotide polymorphism profile was rich in matK &ITS sequencing. Both primers developed a reliable and highly efficient way of determining nucleotide sequence diversity in E.elatum. ITS & matK met the ideal DNA barcode characteristics laid by Consortium of Barcode of Life and hence can be endorsed as ideal barcode markers for the identification of E.elatum in NW Himalayas, India.

Single Nucleotide Polymorphisms in hsp65 and MACPPE12 Genes of Mycobacterium avium subsp. hominissuis

Mycobacterium avium subsp. hominissuis (MAH) are typical inhabitants of the environment, which are known as opportunistic pathogens of animals and humans. The aim of our study was to analyze single-nucleotide polymorphisms (SNPs) in the hsp65 and MACPPE12 genes to characterize the Russian population of MAH in the context of studying phylogenetic relationships and the evolution of geographically distant populations of M. avium subsp. hominissuis. The sequence analysis of the hsp65 and MACPPE12 genes was applied for 40 MAH strains isolated from humans (patients with mycobacteriosis). The nucleotide sequences were aligned to the reference genome of M. avium subsp. hominissuis 104 (accession no. NC_008595.1). The mutational profiles of Russian strains were compared with those isolated in other countries. In total, 40 MAH strains were classified into three different hsp65 sequevars: code 1, code 2, and code 3. The majority of MAH strains (72.5%) belonged to code 1, the same sequevar as for MAH strain 104. The sequence analysis of the MACPPE12 gene revealed 20 SNPs grouped into nine sequevars at the nucleic acid level: NA01, NA02, NA03, NA06, NA10, NA13, NA14, NA19, and NA_Rus01. Eight out of 20 SNPs were nonsynonymous, resulting in seven sequevars at the amino acid level: AA01, AA02, AA04, AA07, AA08, AA13, and AA_Rus01. The sequevar AA02 consisted of three different NA variants with synonymous SNPs profiles: NA02, NA03, and NA06. Half of the MAH strains belonged to the sequevar AA02 (type NA02). The predominant cluster AA02 (type NA02)/code 1 and the unique variant AA_Rus01 (NA_Rus01) were identified among MAH strains from Russia. Thus, we confirmed the relative conservativeness of the nucleotide sequence of the hsp65 gene but the polymorphism of the MACPPE12 gene. At the same time, a comparative analysis of the SNPs profiles of the hsp65 and MACPPE12 genes allowed us to identify differences and similarities between geographically distant populations of MAH, which highlighted the variability of the global population of M. avium species.